INTERNATIONAL JOURNAL OF ONCOLOGY 45: 558-566, 2014
558
Daurinol, a catalytic inhibitor of topoisomerase IIα, suppresses SNU-840 ovarian cancer cell proliferation through cell cycle arrest in S phase KYUNGSU KANG1*, CHU WON NHO1*, NAM DOO KIM2, DAE-GEUN SONG1, YOUNG GYUN PARK1, MINKYUN KIM4, CHEOL-HO PAN1, DONGYUN SHIN3, SEUNG HYUN OH3 and HO-SUK OH5 1
Functional Food Center, Korea Institute of Science and Technology, Gangneung; 2New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu; 3College of Pharmacy, Gachon University, Incheon; 4 Department of Agricultural Biotechnology, Seoul National University, Seoul; 5 Department of Internal Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Republic of Korea Received January 28, 2014; Accepted March 29, 2014 DOI: 10.3892/ijo.2014.2442
Abstract. Daurinol, a lignan from the ethnopharmacological plant Haplophyllum dauricum, was recently reported to be a novel topoisomerase II inhibitor and an alternative to the clinical anticancer agent etoposide based on a colorectal cancer model. In the present study, we elucidated the detailed biochemical mechanism underlying the inhibition of human topoisomerase IIα by daurinol based on a molecular docking study and in vitro biochemical experiments. The computational simulation predicted that daurinol binds to the ATP-binding pocket of topoisomerase IIα. In a biochemical assay, daurinol (10-100 µM) inhibited the catalytic activity of topoisomerase IIα in an ATP concentration-dependent manner and suppressed the ATP hydrolysis activity of the enzyme. However, daurinol did not inhibit topoisomerase I activity, most likely because topoisomerase I does not contain an ATP-binding domain. We also evaluated the anti-proliferative activity of daurinol in ovarian, small cell lung and testicular cancer cells, common target cancers treated with etoposide. Daurinol potently inhibited SNU-840 human ovarian cancer cell proliferation through cell cycle arrest in S phase, while etoposide induced G2/M phase arrest. Daurinol induced the increased expression of cyclin E, cyclin A and E2F-1, which are important proteins regulating S phase initiation and progression. Daurinol did not induce abnormal cell and nuclear
Correspondence to: Professor Ho-Suk Oh, Department of Internal Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, 415 Bangdong-ri, Gangneung-si, Gangwon-do 210-711, Republic of Korea E-mail: [email protected] *
Contributed equally
Key words: daurinol, topoisomerase IIα inhibitor, ovarian cancer, ATP binding pocket, secondary leukemia
enlargement in SNU-840 cells, in contrast to etoposide. Based on these data, we suggest that daurinol is a potential anticancer drug candidate for the treatment of human ovarian cancer with few side effects. Introduction Topoisomerases are essential enzymes in all organisms that are involved in the topological homeostasis of DNA molecules during DNA replication, transcription and chromosomal segregation. Topoisomerases are classified into two classes, topoisomerase I and II, which create single and double-strand breaks, respectively. Because topoisomerases are crucial enzymes in DNA replication, they have served as primary targets for the development of anticancer agents (1,2). Topoisomerase II inhibitors such as etoposide, teniposide and doxorubicin have been extensively used in clinical cancer treatment. However, these agents also have undesirable side effects, including immunosuppression, myelosuppression, gastrointestinal toxicity and the development of secondary leukemia (3). Thus, the discovery of new topoiso merase inhibitors with fewer side effects from natural products has received a great deal of attention (4,5). Daurinol is a natural arylnaphthalene lignan isolated from the traditional medicinal plant Haplophyllum dauricum (6). According to an ethnopharmacological study, this plant has been used to treat tumors in Russia (7). The chemical structure of daurinol is similar to that of the clinical anticancer agent VP-16 (also known as etoposide phosphate). Recently, we suggested that daurinol could be a promising antitumor agent with minimal side effects, compared to etoposide, based on in vitro and in vivo results. Daurinol suppressed the growth of human colorectal cancer cells through the inhibition of human topoisomerase IIα in vitro and dramatically inhibited the growth of HCT116 tumors in a nude mouse xenograft model. Moreover, daurinol did not show severe side effects such as loss of body weight and hematological toxicity, i.e., loss of white blood cells and red blood cells, and decreased hemoglobin content (5).
KANG et al: DAURINOL INHIBITS OVARIAN CANCER VIA TOPOISOMERASE II INHIBITION
However, this previous study revealed some limitations of daurinol that must be resolved to develop this agent as a novel alternative to etoposide in clinical trials. First, the availability of natural daurinol from the plant H. dauricum is limited because its content is quite low (0.013% of the plant dry weight) (6). Second, daurinol inhibits human topoiso merase IIα activity in the millimolar range (5). We neither elucidated the inhibitory activity against other topoisomerases, including topoisomerase I, nor identified the detailed biochemical mechanism underlying the daurinol-induced inhibition of topoisomerase IIα. Last but most important, we did not examine its effect on other cancer cell types besides colorectal cancer, even though etoposide is also frequently used to treat other cancers such as ovarian, small-cell lung and testicular cancer (3,8). To address these limitations, in the present study, we used synthetic daurinol prepared by a regioselective chemical synthesis. In addition, we have proposed a biochemical mechanism underlying the inhibitory action of daurinol against human topoisomerase IIα based on a computational molecular docking study and biochemical experiments. Finally, we tested the anticancer activity of synthetic daurinol against human ovarian, small-cell lung and testicular cancer cells and investi gated the effects of daurinol on cell cycle distribution and cell morphology in the selected cancer cells in comparison to etoposide. Materials and methods Reagents. Daurinol used in this study was chemically synthesized according to recently reported methods (9). Natural daurinol was isolated from Haplophyllum dauricum as previously described (6). Dimethyl sulfoxide (DMSO), etoposide, novobiocin, propidium iodide (PI) and ATP disodium salt were purchased from Sigma (St. Louis, MO, USA). Camptothecin was purchased from TopoGEN, Inc. (Port Orange, FL, USA). Daurinol, etoposide and camptothecin were dissolved in DMSO for cellular treatment. Antibodies against E2F-1 (3742), cyclin A (4656) and Cdk4 (2906) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Cdk2 (sc-163), cyclin E (sc-247), cyclin D1 (sc-753) and β -actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary anti-rabbit and anti-mouse antibodies were purchased from Santa Cruz Biotechnology. Molecular docking analysis. The crystal structure of human topoisomerase IIα (ATP2ATPase) complexed with AMPPNP (a non-hydrolyzable ATP analog) from the Protein Data Bank (PDB code 1ZXM) was used for the docking simulation. Daurinol was built using the Maestro build panel and minimized using the impact module of Maestro in the Schrödinger suite program. The starting coordinates of the ATP2ATPASE were further modified for the prediction of daurinol binding. The protein structure was minimized using the Protein Preparation Wizard by applying an OPLS force field. For grid generation, the binding site was defined as the centroid of the AMPPNP. Ligand docking into the active site of ATP2ATPASE was performed using the Schrödinger docking program Glide (Schrödinger, Inc., USA). Energy-minimized daurinol was
559
docked into the prepared receptor grid. The best-docked poses were selected based on the lowest Glide scores. The molecular graphics of the inhibitor-binding pocket and refined docking model for daurinol were generated using the PyMol package (http://www.pymol.org). Measurement of human topoisomerase IIα catalytic activity. The inhibitory activity of daurinol against human topoiso merase IIα was measured using the Topoisomerase II Drug Screening kit (TopoGEN, Inc.). The standard reaction mixture (20 µl) contained 250 ng of supercoiled DNA (pHOT1), 0.7 µl of topoisomerase IIα, and different concentrations of ATP (0.5, 1 or 2 mM) and the tested compounds dissolved in DMSO. The final concentration of DMSO was 1%, and the topo isomerase II poison etoposide was used as a positive control. Reactions were initiated by the addition of the supercoiled DNA substrate. The reaction mixture was incubated at 37˚C for 30 min. Other procedures were performed as described previously (5). Measurement of human topoisomerase I catalytic activity. The inhibitory activity of daurinol against human topoiso merase I was measured using the Topoisomerase I Assay kit (TopoGEN, Inc.). The standard reaction mixture (20 µl) contained 10 mM Tris-HCl (pH 7.9), 1 mM EDTA, 150 mM NaCl, 0.1% bovine serum albumin (BSA), 0.1 mM spermidine, 5% glycerol, 250 ng of supercoiled DNA (pHOT1), 0.7 µl of topoisomerase I, and the test compound dissolved in DMSO. The final concentration of DMSO was 1%, and the topoisomerase I poison camptothecin was used as a positive control. Reactions were initiated by the addition of the supercoiled DNA substrate. The reaction mixture was incubated at 37˚C for 30 min, and 2 µl of 10% sodium dodecyl sulfate was added to stop the reaction. Additional procedures were performed using procedures similar to the topoisomerase II assay, as described previously (5). ATPase activity assay. The DNA-dependent ATP hydrolysis activity of human topoisomerase IIα was determined by quanti fying hydrolyzed inorganic phosphate using the malachite green assay with slight modifications (10‑12). Briefly, the standard reaction mixture (20 µl) contained 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 30 µg/ml BSA, 200 ng of supercoiled DNA (pHOT-1), 2.0 µl of human topoisomerase IIα (TopoGEN, Inc.); and 100 µM daurinol or 400 µM novobiocin dissolved in DMSO. The final concentration of DMSO was 1%, and novobiocin was used as a positive control (12,13). The reaction was initiated by adding ATP at a final concentration of 2 mM (Sigma) and was incubated at 37˚C for 30 min. The reaction was stopped by the addition of the Lanzetta reagent (80 µl, freshly prepared mixture of 0.035% malachite green-HCl and 4.2% ammonium molybdate in 4 N HCl at a ratio of 3:1 and 0.2% CHAPS), and the color development was read immediately using a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA) at 620 nm. The amount of inorganic phosphate was calculated using a phosphate (potassium dihydrogen phosphate, KH2PO3) standard curve. Enzyme activity was expressed as µM phosphate produced per min reaction.
560
INTERNATIONAL JOURNAL OF ONCOLOGY 45: 558-566, 2014
Cell culture. SK-OV-3 and NIH:OVCAR-3 human ovarian cancer; and NTERA-2 cl.D1 (NT2-D1), NCCIT human testicular cancer cell lines were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA). The SNU-840 human ovarian cancer; and NCI-H69, NCI-H146, NCI-H187 and NCI-H417 human small-cell lung cancer cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). These cells were cultured in RPMI‑1640 supplemented with 25 mM HEPES, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at sub-confluence in a 95% air and 5% CO2 humidified atmosphere at 37˚C. Measurement of anti-proliferative activity. Cancer cells (5x103 cells/well) were plated in 96‑well plates, incubated at 37˚C for 24 h, and treated with daurinol for 48 h. Cell viability was determined using the EZ-Cytox cell viability assay kit (Daeil Lab Service, Ltd., Seoul), as previously described (14). Cell cycle analysis. NIH:OVCAR-3 (5x10 5 cells/well), SNU-840 (3x105 cells/well) and NCCIT (3x105 cells/well) cells were plated in 6‑well plates, incubated at 37˚C for 24 h, and treated with daurinol or etoposide for 24 and 48 h. The cells were stained with PI, and their DNA contents were analyzed using a FACSCalibur flow cytometer (BectonDickinson, San Jose, CA, USA) and Modfit LT V3.0 software (Verity Software House, Topsham, ME, USA) as previously described (5). Western blot analysis. SNU-840 cells (5x105) were seeded on 60‑mm dishes, incubated for 24 h, and then treated with daurinol for 24 and 48 h. Additional procedures were performed as previously described (15). The relative protein expression was measured by densitometry. Evaluation of cell and nuclear size. To evaluate the effect of daurinol and etoposide on cell and nuclear size, we performed fluorescence pulse signal analysis of PI-stained SNU-840 cells as previously described (15,16). The treatment and flow cytometric DNA content analysis were performed as described above in the section of cell cycle analysis. FSC-H and FL2-W values, which correlate with cell and nuclear size, respectively, were measured using flow cytometry. The mean values of FSC-H and FL2-W were calculated using the histogram stati stics tool from CellQuest Pro software (Becton-Dickinson). To compare the FSC-H and FL2-W distributions of the control and chemical-treated cells, we used the Kolmogorov-Smirnov statistics tools from CellQuest Pro software. We also observed SNU-840 cell morphology after treatment with daurinol or etoposide using an Olympus CK40 phase contrast microscope (Tokyo, Japan). Statistical analysis. The values represent the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 5 software (La Jolla, CA, USA). A paired two-tailed Student's t-test was used to compare the IC50 values of daurinol and etoposide in each cell line. One-way analysis of variance (ANOVA) followed by Dunnett's or Tukey's multiple comparison test was used to analyze all other data. P
558
Daurinol, a catalytic inhibitor of topoisomerase IIα, suppresses SNU-840 ovarian cancer cell proliferation through cell cycle arrest in S phase KYUNGSU KANG1*, CHU WON NHO1*, NAM DOO KIM2, DAE-GEUN SONG1, YOUNG GYUN PARK1, MINKYUN KIM4, CHEOL-HO PAN1, DONGYUN SHIN3, SEUNG HYUN OH3 and HO-SUK OH5 1
Functional Food Center, Korea Institute of Science and Technology, Gangneung; 2New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu; 3College of Pharmacy, Gachon University, Incheon; 4 Department of Agricultural Biotechnology, Seoul National University, Seoul; 5 Department of Internal Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Republic of Korea Received January 28, 2014; Accepted March 29, 2014 DOI: 10.3892/ijo.2014.2442
Abstract. Daurinol, a lignan from the ethnopharmacological plant Haplophyllum dauricum, was recently reported to be a novel topoisomerase II inhibitor and an alternative to the clinical anticancer agent etoposide based on a colorectal cancer model. In the present study, we elucidated the detailed biochemical mechanism underlying the inhibition of human topoisomerase IIα by daurinol based on a molecular docking study and in vitro biochemical experiments. The computational simulation predicted that daurinol binds to the ATP-binding pocket of topoisomerase IIα. In a biochemical assay, daurinol (10-100 µM) inhibited the catalytic activity of topoisomerase IIα in an ATP concentration-dependent manner and suppressed the ATP hydrolysis activity of the enzyme. However, daurinol did not inhibit topoisomerase I activity, most likely because topoisomerase I does not contain an ATP-binding domain. We also evaluated the anti-proliferative activity of daurinol in ovarian, small cell lung and testicular cancer cells, common target cancers treated with etoposide. Daurinol potently inhibited SNU-840 human ovarian cancer cell proliferation through cell cycle arrest in S phase, while etoposide induced G2/M phase arrest. Daurinol induced the increased expression of cyclin E, cyclin A and E2F-1, which are important proteins regulating S phase initiation and progression. Daurinol did not induce abnormal cell and nuclear
Correspondence to: Professor Ho-Suk Oh, Department of Internal Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, 415 Bangdong-ri, Gangneung-si, Gangwon-do 210-711, Republic of Korea E-mail: [email protected] *
Contributed equally
Key words: daurinol, topoisomerase IIα inhibitor, ovarian cancer, ATP binding pocket, secondary leukemia
enlargement in SNU-840 cells, in contrast to etoposide. Based on these data, we suggest that daurinol is a potential anticancer drug candidate for the treatment of human ovarian cancer with few side effects. Introduction Topoisomerases are essential enzymes in all organisms that are involved in the topological homeostasis of DNA molecules during DNA replication, transcription and chromosomal segregation. Topoisomerases are classified into two classes, topoisomerase I and II, which create single and double-strand breaks, respectively. Because topoisomerases are crucial enzymes in DNA replication, they have served as primary targets for the development of anticancer agents (1,2). Topoisomerase II inhibitors such as etoposide, teniposide and doxorubicin have been extensively used in clinical cancer treatment. However, these agents also have undesirable side effects, including immunosuppression, myelosuppression, gastrointestinal toxicity and the development of secondary leukemia (3). Thus, the discovery of new topoiso merase inhibitors with fewer side effects from natural products has received a great deal of attention (4,5). Daurinol is a natural arylnaphthalene lignan isolated from the traditional medicinal plant Haplophyllum dauricum (6). According to an ethnopharmacological study, this plant has been used to treat tumors in Russia (7). The chemical structure of daurinol is similar to that of the clinical anticancer agent VP-16 (also known as etoposide phosphate). Recently, we suggested that daurinol could be a promising antitumor agent with minimal side effects, compared to etoposide, based on in vitro and in vivo results. Daurinol suppressed the growth of human colorectal cancer cells through the inhibition of human topoisomerase IIα in vitro and dramatically inhibited the growth of HCT116 tumors in a nude mouse xenograft model. Moreover, daurinol did not show severe side effects such as loss of body weight and hematological toxicity, i.e., loss of white blood cells and red blood cells, and decreased hemoglobin content (5).
KANG et al: DAURINOL INHIBITS OVARIAN CANCER VIA TOPOISOMERASE II INHIBITION
However, this previous study revealed some limitations of daurinol that must be resolved to develop this agent as a novel alternative to etoposide in clinical trials. First, the availability of natural daurinol from the plant H. dauricum is limited because its content is quite low (0.013% of the plant dry weight) (6). Second, daurinol inhibits human topoiso merase IIα activity in the millimolar range (5). We neither elucidated the inhibitory activity against other topoisomerases, including topoisomerase I, nor identified the detailed biochemical mechanism underlying the daurinol-induced inhibition of topoisomerase IIα. Last but most important, we did not examine its effect on other cancer cell types besides colorectal cancer, even though etoposide is also frequently used to treat other cancers such as ovarian, small-cell lung and testicular cancer (3,8). To address these limitations, in the present study, we used synthetic daurinol prepared by a regioselective chemical synthesis. In addition, we have proposed a biochemical mechanism underlying the inhibitory action of daurinol against human topoisomerase IIα based on a computational molecular docking study and biochemical experiments. Finally, we tested the anticancer activity of synthetic daurinol against human ovarian, small-cell lung and testicular cancer cells and investi gated the effects of daurinol on cell cycle distribution and cell morphology in the selected cancer cells in comparison to etoposide. Materials and methods Reagents. Daurinol used in this study was chemically synthesized according to recently reported methods (9). Natural daurinol was isolated from Haplophyllum dauricum as previously described (6). Dimethyl sulfoxide (DMSO), etoposide, novobiocin, propidium iodide (PI) and ATP disodium salt were purchased from Sigma (St. Louis, MO, USA). Camptothecin was purchased from TopoGEN, Inc. (Port Orange, FL, USA). Daurinol, etoposide and camptothecin were dissolved in DMSO for cellular treatment. Antibodies against E2F-1 (3742), cyclin A (4656) and Cdk4 (2906) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against Cdk2 (sc-163), cyclin E (sc-247), cyclin D1 (sc-753) and β -actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary anti-rabbit and anti-mouse antibodies were purchased from Santa Cruz Biotechnology. Molecular docking analysis. The crystal structure of human topoisomerase IIα (ATP2ATPase) complexed with AMPPNP (a non-hydrolyzable ATP analog) from the Protein Data Bank (PDB code 1ZXM) was used for the docking simulation. Daurinol was built using the Maestro build panel and minimized using the impact module of Maestro in the Schrödinger suite program. The starting coordinates of the ATP2ATPASE were further modified for the prediction of daurinol binding. The protein structure was minimized using the Protein Preparation Wizard by applying an OPLS force field. For grid generation, the binding site was defined as the centroid of the AMPPNP. Ligand docking into the active site of ATP2ATPASE was performed using the Schrödinger docking program Glide (Schrödinger, Inc., USA). Energy-minimized daurinol was
559
docked into the prepared receptor grid. The best-docked poses were selected based on the lowest Glide scores. The molecular graphics of the inhibitor-binding pocket and refined docking model for daurinol were generated using the PyMol package (http://www.pymol.org). Measurement of human topoisomerase IIα catalytic activity. The inhibitory activity of daurinol against human topoiso merase IIα was measured using the Topoisomerase II Drug Screening kit (TopoGEN, Inc.). The standard reaction mixture (20 µl) contained 250 ng of supercoiled DNA (pHOT1), 0.7 µl of topoisomerase IIα, and different concentrations of ATP (0.5, 1 or 2 mM) and the tested compounds dissolved in DMSO. The final concentration of DMSO was 1%, and the topo isomerase II poison etoposide was used as a positive control. Reactions were initiated by the addition of the supercoiled DNA substrate. The reaction mixture was incubated at 37˚C for 30 min. Other procedures were performed as described previously (5). Measurement of human topoisomerase I catalytic activity. The inhibitory activity of daurinol against human topoiso merase I was measured using the Topoisomerase I Assay kit (TopoGEN, Inc.). The standard reaction mixture (20 µl) contained 10 mM Tris-HCl (pH 7.9), 1 mM EDTA, 150 mM NaCl, 0.1% bovine serum albumin (BSA), 0.1 mM spermidine, 5% glycerol, 250 ng of supercoiled DNA (pHOT1), 0.7 µl of topoisomerase I, and the test compound dissolved in DMSO. The final concentration of DMSO was 1%, and the topoisomerase I poison camptothecin was used as a positive control. Reactions were initiated by the addition of the supercoiled DNA substrate. The reaction mixture was incubated at 37˚C for 30 min, and 2 µl of 10% sodium dodecyl sulfate was added to stop the reaction. Additional procedures were performed using procedures similar to the topoisomerase II assay, as described previously (5). ATPase activity assay. The DNA-dependent ATP hydrolysis activity of human topoisomerase IIα was determined by quanti fying hydrolyzed inorganic phosphate using the malachite green assay with slight modifications (10‑12). Briefly, the standard reaction mixture (20 µl) contained 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 30 µg/ml BSA, 200 ng of supercoiled DNA (pHOT-1), 2.0 µl of human topoisomerase IIα (TopoGEN, Inc.); and 100 µM daurinol or 400 µM novobiocin dissolved in DMSO. The final concentration of DMSO was 1%, and novobiocin was used as a positive control (12,13). The reaction was initiated by adding ATP at a final concentration of 2 mM (Sigma) and was incubated at 37˚C for 30 min. The reaction was stopped by the addition of the Lanzetta reagent (80 µl, freshly prepared mixture of 0.035% malachite green-HCl and 4.2% ammonium molybdate in 4 N HCl at a ratio of 3:1 and 0.2% CHAPS), and the color development was read immediately using a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA) at 620 nm. The amount of inorganic phosphate was calculated using a phosphate (potassium dihydrogen phosphate, KH2PO3) standard curve. Enzyme activity was expressed as µM phosphate produced per min reaction.
560
INTERNATIONAL JOURNAL OF ONCOLOGY 45: 558-566, 2014
Cell culture. SK-OV-3 and NIH:OVCAR-3 human ovarian cancer; and NTERA-2 cl.D1 (NT2-D1), NCCIT human testicular cancer cell lines were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA). The SNU-840 human ovarian cancer; and NCI-H69, NCI-H146, NCI-H187 and NCI-H417 human small-cell lung cancer cell lines were obtained from the Korean Cell Line Bank (Seoul, Korea). These cells were cultured in RPMI‑1640 supplemented with 25 mM HEPES, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at sub-confluence in a 95% air and 5% CO2 humidified atmosphere at 37˚C. Measurement of anti-proliferative activity. Cancer cells (5x103 cells/well) were plated in 96‑well plates, incubated at 37˚C for 24 h, and treated with daurinol for 48 h. Cell viability was determined using the EZ-Cytox cell viability assay kit (Daeil Lab Service, Ltd., Seoul), as previously described (14). Cell cycle analysis. NIH:OVCAR-3 (5x10 5 cells/well), SNU-840 (3x105 cells/well) and NCCIT (3x105 cells/well) cells were plated in 6‑well plates, incubated at 37˚C for 24 h, and treated with daurinol or etoposide for 24 and 48 h. The cells were stained with PI, and their DNA contents were analyzed using a FACSCalibur flow cytometer (BectonDickinson, San Jose, CA, USA) and Modfit LT V3.0 software (Verity Software House, Topsham, ME, USA) as previously described (5). Western blot analysis. SNU-840 cells (5x105) were seeded on 60‑mm dishes, incubated for 24 h, and then treated with daurinol for 24 and 48 h. Additional procedures were performed as previously described (15). The relative protein expression was measured by densitometry. Evaluation of cell and nuclear size. To evaluate the effect of daurinol and etoposide on cell and nuclear size, we performed fluorescence pulse signal analysis of PI-stained SNU-840 cells as previously described (15,16). The treatment and flow cytometric DNA content analysis were performed as described above in the section of cell cycle analysis. FSC-H and FL2-W values, which correlate with cell and nuclear size, respectively, were measured using flow cytometry. The mean values of FSC-H and FL2-W were calculated using the histogram stati stics tool from CellQuest Pro software (Becton-Dickinson). To compare the FSC-H and FL2-W distributions of the control and chemical-treated cells, we used the Kolmogorov-Smirnov statistics tools from CellQuest Pro software. We also observed SNU-840 cell morphology after treatment with daurinol or etoposide using an Olympus CK40 phase contrast microscope (Tokyo, Japan). Statistical analysis. The values represent the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 5 software (La Jolla, CA, USA). A paired two-tailed Student's t-test was used to compare the IC50 values of daurinol and etoposide in each cell line. One-way analysis of variance (ANOVA) followed by Dunnett's or Tukey's multiple comparison test was used to analyze all other data. P
